Transcription factor having zinc finger domain

ABSTRACT

Using an undifferentiated mouse CL6 cell line, DMSO was added to induce its differentiation into cardiac muscular cells in order to obtain gene fragments whose expression elevated upon the induction. The isolated gene had zinc finger domains and showed a significant homology to the Sp1 family genes. Furthermore, a human gene corresponding to this mouse gene was isolated. The protein encoded by this gene existed in the nucleus and bonded to a GC-box. The protein was revealed to repress the transcription regulatory activity of the CMV promoter and thus serves as a transcription factor.

Reference To Sequence Listing Submitted Via EFS-WEB

The entire content of the following electronic submission of thesequence listing via the USPTO EFS-WEB server, as authorized and setforth in MPEP § 1730 II.B.2(a)(C), is incorporated herein by referencein its entirety for all purposes. The sequence listing is identified onthe electronically filed text file as follows:

File Name Date of Creation Size (bytes) 532842000500Seqlist.txt Jan. 29,2008 30,474 bytes

TECHNICAL FIELD

The present invention relates to novel proteins with zinc finger domainsthat bind to a GC-box, genes encoding these proteins, as well asproduction and use of these proteins and their genes.

BACKGROUND ART

G-rich elements, such as GC-box or GT-box, are sequences that bind totranscription factors which have been found in promoter sequences ofvarious genes including house keeping genes as well as tissue specificgenes, parentally expressing genes, etc. The G-rich elements are closelyrelated to development and differentiation, in addition to geneexpression upon the binding of transcription factors.

Sp1 has been found as a DNA-binding transcription factor that binds tothe GC-box of the SV-40 promoter sequence to regulate thetranscriptional activity of the promoter (Dynan W S. et al. (1983) Cell35, 79-87; Gidoni D. et al. (1984) Nature 312, 409-13). The Sp1 hasthree zinc finger domains in its C-terminal region and binds to aGC/GT-box through the domains (Kadonaga J T. et al. (1987) Cell 51,1079-90). Moreover, it has been known that the Sp1 has twoglutamine-rich regions required for the activation of transcription(Courey A J. et al. (1988) Cell 55, 887-98), and yet twoserine/threonine-rich regions. Transcription factor homologues, Sp2(Kingsley C. et al. (1992) Mol Cell Biol 12, 4251-61), Sp3, and Sp4(Hagen G. et al. (1992) Nucleic Acids Res 20, 5519-25), with a highhomology to the Sp1 are known to exist and they comprise a family. Eachtranscription factor has three highly conserved zinc finger domains inits C-terminal region, and besides, a glutamine-rich region and/or aserine/threonine-rich region. While the Sp3 and Sp4, similarly to theSp1, bind to a GC/GT-box (Hagen G. et al. (1992) Nucleic Acids Res. 20,5519-25; Hagen G. et al. (1994) EMBO J 13, 3843-51), the Sp2 is reportednot to bind to a GC-box and rather binds to a GT-box in the Vα promoterregion of TCR (Kingsley C. et al. (1992) Mol. Cell. Biol. 12, 4251-61).

Recently, many transcription factors with high homology to the threezinc finger domains of Sp1 have been reported (Philipsen S. et al.(1999) Nucleic Acids Res. 27, 2991-3000). As in the case with Sp1, theyall have been shown to have three zinc finger domains in its C-terminalregions and bind to a GC/GT-box; however, other regions share only a lowhomology with the Sp-family and do not have a glutamine-rich region thatfunctions as a transcription activation region in Sp1.

These GC/GT-box binding transcription factors have been known to enhanceor repress the transcriptional activity, and thus, are considered toregulate gene expression in various cells during development,differentiation, and such. Such characteristics of the GC/GT-box bindingtranscription factors described above presently make them remarkable astargets in developing therapeutic agents.

DISCLOSURE OF THE INVENTION

The present invention provides novel proteins with zinc finger domainsthat bind to a GC-box, genes encoding the proteins, and moleculesfunctionally equivalent thereto, as well as production and use thereof.

The present inventors searched for genes whose expression increases inresponse to the differentiation of the mouse CL6 cell, for which asystem to induce differentiation to cardiomyocyte-like cell by DMSOaddition has been established, to discover genes involved in cardiacmuscle differentiation.

First, the present inventors conducted the subtraction method usingpolyA+ RNA prepared from undifferentiated CL6 cells and those 4 daysafter DMSO addition, and obtained many gene fragments whose expressionhad been increased upon the induction of differentiation. One cloneamong them, clone PS40-285, was revealed to be a novel gene with zincfinger domains by a homology search and amino acid sequence motifsearch. Accordingly, cloning of a cDNA comprising the whole ORF of thenovel gene was carried out and its nucleotide sequence was determined.

As a result, the protein encoded by the novel murine gene (m285) wasidentified as a protein consisting of 398 amino acids with three zincfinger motifs. These three zinc finger domains were located near itsC-terminus and the sequence showed high homology with the three zincfinger domains of known SP family transcription factors (SP1, 2, 3, and4). Further, a region near the N-terminus was also found to share a highhomology with SP1, 2, and 4. Other regions of the protein showed no highhomology with known genes.

Next, m285 gene expression during the differentiation-inducing processof CL6 cells by DMSO stimulation was examined to reveal a transientexpression of the gene 4 days after the induction of differentiation.Moreover, tissue distribution analysis of the gene expression by theNorthern blot analysis revealed that m285 is very highly expressed in 17days postcoitum (dpc) murine embryo and almost no expression could beobserved in 7 dpc, 11 dpc, and 15 dpc embryos. However, in adulttissues, expression of the gene in testicular tissue was observed, butit was weakly expressed in brain, heart, liver, and kidney.

The present inventors also cloned a cDNA containing the total ORF of thenovel human gene (h285), which is the human counterpart of m285. Theh285 encoded 398 amino acids, which is the same as m285. According tothe comparison of the obtained h285 to m285, it had a homology of 91.1%at the nucleotide sequence level within the coding region, and 97.5%homology at the amino acid sequence level.

Transcription factors are generally localized in the nucleus. Thus, thecellular distribution of m285 was analyzed. As a result, m285 wasrevealed to exist in the nucleus and the C-terminal region includingthree zinc finger domains was required for its nuclear translocation.Moreover, according to a DNA binding assay, m285 was revealed to bind tothe nucleotide sequence of a GC-box, identical to the SP familytranscription factors, at its C-terminal region that include the threezinc finger domains. The GC-box is known to exist in thepromoter/enhancer regions of many genes. According to these facts, gene285 is expected to regulate the transcription of genes through thebinding to a GC-box.

Then the mode of transcriptional regulation (enhancement or repressionof transcription) of 285 was examined by the mammalian One-HybridSystem. As a result, 285 was revealed to repress the transcriptionalactivity of the CMV promoter. Thus, the function of the 285 as atranscription factor was revealed.

The present invention was accomplished based on the knowledge describedabove, which provides novel proteins “285” having zinc finger domainsthat bind to a GC-box, genes encoding the proteins, and moleculesfunctionally equivalent thereto, as well as production and use thereof.

More specifically, the present invention provides:

-   -   (1) a DNA selected from the group consisting of:

-   (a) a DNA encoding a protein comprising the amino acid sequence of    SEQ ID NO: 2 or 4;

-   (b) a DNA comprising the coding region of the nucleotide sequence of    SEQ ID NO: 1 or 3;

-   (c) a DNA encoding a protein comprising the amino acid sequence of    SEQ ID NO: 2 or 4 in which one or more amino acids are substituted,    deleted, inserted, and/or added, and wherein the protein is    functionally equivalent to the protein consisting of the amino acid    sequence of SEQ ID NO: 2 or 4; and

-   (d) a DNA hybridizing under stringent conditions with a DNA    consisting of the nucleotide sequence of SEQ ID NO: 1 or 3 which    encodes a protein functionally equivalent to the protein consisting    of the amino acid sequence of SEQ ID NO: 2 or 4;    -   (2) a DNA encoding a partial peptide of the protein consisting        of the amino acid sequence of SEQ ID NO: 2 or 4;    -   (3) a protein or a peptide encoded by the DNA of (1) or (2);    -   (4) a vector into which the DNA of (1) or (2) is inserted;    -   (5) a transformed cell retaining the DNA of (1) or (2), or the        vector of (4);    -   (6) a method for producing the protein or peptide of (3),        wherein the method comprises the steps of: culturing the host        cell of (5), and recovering the expressed protein from said host        cell or the culture supernatant thereof;    -   (7) an antibody binding to the protein of (3);    -   (8) a polynucleotide comprising at least 15 nucleotides which is        complementary to either a DNA consisting of the nucleotide        sequence of SEQ ID NO: 1 or 3, or the complementary strand        thereof;    -   (9) a method of screening for a compound that binds to the        protein of (3), wherein the method comprises the steps of:

-   (a) contacting said protein or partial peptide thereof with a test    sample;

-   (b) detecting the binding activity of said protein or partial    peptide thereof with the test sample; and

-   (c) selecting the compound that binds to said protein or partial    peptide thereof;    -   (10) a method of screening for a compound that controls the        transcriptional regulation activity of the protein of (3),        wherein the method comprises the steps of:

-   (a) contacting said protein or partial peptide thereof with a test    sample;

-   (b) detecting the transcriptional regulation activity of said    protein or partial peptide thereof; and

-   (c) selecting the compound that decreases or increases the    transcriptional regulation activity detected in (b) compared with    that observed in the absence of the test sample;    -   (11) a compound that can be isolated by the screening method        of (9) or (10);    -   (12) a promoter activity regulator comprising the protein or        peptide of (3), or a DNA encoding them; and    -   (13) a promoter activity regulator of (12) wherein the promoter        is the CMV promoter.

The present invention provides a murine derived gene “m285” and humanderived gene “h285”, which encode novel proteins with zinc fingerdomains that bind to a GC-box; hereinafter, “m285” and “h285” arecollectively designated as “285” if necessary.

The nucleotide sequences of m285 and h285 are shown in SEQ ID NO: 1 and3, respectively. The amino acid sequences of the proteins encoded bym285 and h285 are shown in SEQ ID NO: 2 and 4, respectively.

m285 gene of the present invention was isolated as a novel gene whoseexpression increases shortly after the induction of differentiation byadding DMSO to undifferentiated mouse CL6 cells. On the other hand, theh285 gene of the present invention was isolated as a homologue of them285 of human. According to the result of homology analysis, theproteins encoded by the genes of the present invention comprise threezinc finger domains, which showed high homology with those of the Spfamily transcription factors. Further, the proteins were considered tofunction as a transcription factor due to its distribution in the cellnucleus, and binding by recognizing the nucleotide sequence of a GC-box.Actually, the 285 proteins of the present invention have the function ofrepressing the CMV promoter activity in MG63 cells. Considering itsexpression and structural property, the 285 proteins of the presentinvention were expected to be molecules with an important function invivo, and thus, serve as practical targets for the development oftherapeutic agents. Moreover, the 285 proteins of the present invention,partial peptides thereof, and DNAs encoding them, may be utilized as adrug (reagent and therapeutic agent) that represses the activity of apromoter, such as the CMV promoter.

The present invention includes proteins functionally equivalent to the285 proteins (proteins consisting of the amino acid sequences of SEQ IDNO: 2 or 4). Such proteins include, for example, mutants and homologuesof the 285 proteins. Herein, the term “functionally equivalent”indicates that the protein of interest, identical with the 285 proteins,functions as a transcription factor with zinc finger domain(s).Specifically, such functions include, for example, binding to a GC-boxthrough zinc finger domain(s), repression of the CMV promoter activityin MG63 cells, and so on.

A binding activity of a protein of interest to a GC-box can bedetermined, for example, by the gel shift assay as described inExamples. Specifically, after mixing an objective protein and labeledDNA probes comprising a GC-box, nondenaturing polyacrylamide gelelectrophoresis is carried out to detect the mobility of the labeled DNAprobes on the gel. When the objective protein binds to the labeled DNAcomprising a GC-box, the mobility of the band of the DNA-protein complexis lower than that of the labeled DNA alone. Therefore, by detecting aband with low mobility, the objective protein can be judged to bind to aGC-box. Alternatively, repression of the CMV promoter activity in MG63cells by an objective protein can be determined by repression assaydescribed in Example 8.

As a method well known to those skilled in the art for preparing aprotein functionally equivalent to a given protein, methods forintroducing mutations into proteins are known. For example, one skilledin the art can prepare proteins functionally equivalent to the 285proteins by introducing an appropriate mutation in the amino acidsequence of the proteins by site-directed mutagenesis (Hashimoto-GotohT. et al. (1995) Gene 152, 271-275; Zoller M J. and Smith M. (1983)Methods Enzymol. 100, 468-500; Kramer W. et al. (1984) Nucleic AcidsRes. 12, 9441-9456;Kramer W. and Fritz H J.(1987) Methods. Enzymol. 154,350-367; Kunkel T A. (1985) Proc. Natl. Acad. Sci. USA. 82, 488-492;Kunkel (1988) Methods Enzymol. 85, 2763-2766). Mutation of amino acidscan occur in nature too. The proteins of the present invention includethose proteins that comprise the amino acid sequences of the 285proteins, wherein one or more amino acids are mutated, yet arefunctionally equivalent to the proteins comprising the sequence of 285proteins. It is considered that the number of amino acids to be mutatedin such a mutant is generally 50 amino acids or less, preferably 30amino acids or less, more preferably 10 amino acids or less (forexample, 5 amino acids or less).

As for the amino acid residue to be mutated, it is preferable that it ismutated into a different amino acid so that the properties of the aminoacid side-chain are conserved. Examples of properties of amino acid sidechains are hydrophobic amino acids (A, I, L, M, F, P, W, Y, V),hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), and aminoacids comprising the following side chains: an aliphatic side-chain (G,A, V, L, I, P); a hydroxyl group containing side-chain (S, T, Y); asulfur atom containing side-chain (C, M); a carboxylic acid and amidecontaining side-chain (D, N, E, Q); a base containing side-chain (R, K,H); and an aromatic containing side-chain (H, F, Y, W) (The parentheticletters indicate the one-letter codes for amino acids).

It is well known that a protein having deletion, addition, and/orsubstitution of one or more amino acid residues in its sequence canretain the original biological activity (Mark D F. et al. (1984) Proc.Natl. Acad. Sci. U.S.A. 81, 5662-5666; Zoller M J. and Smith M. (1982)Nucleic Acids Res. 10, 6487-6500;Wang A. et al. Science 224,1431-1433;Dalbadie-McFarland G. et al. (1982) Proc. Natl. Acad. Sci. U.S.A. 79,6409-6413).

A fusion protein comprising a 285 protein is encompassed in the protein,wherein plural amino acid residues are added to the amino acid sequenceof 285 proteins. Fusion proteins are fusions of these proteins withother peptides or proteins, and are included within the scope of thepresent invention. Fusion proteins can be made by techniques well knownto a person skilled in the art, such as, by linking the DNA encoding a285 protein with DNA encoding other peptides or proteins so that theframes match, inserting this linked-DNA into an expression vector, andexpressing it in a host. There is no restriction as to what peptides orproteins may be fused to a protein of the present invention.

Known peptides, for example, FLAG (Hopp, T. P. et al., Biotechnology(1988) 6, 1204-1210), 6×His consisting of six His (histidine) residues,10×His, Influenzaagglutinin (HA), humanc-myc fragment, VSV-GP fragment,p18HIV fragment, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lcktag, α-tubulin fragment, B-tag, Protein C fragments, etc. can be used aspeptides to be fused to a protein of the present invention. Examples ofproteins that may be fused to a protein of the present inventioninclude, but are not limited to, GST (glutathione-S-transferase),Influenza agglutinin (HA), immunoglobulin constant region,β-galactosidase, MBP (maltose-binding protein), etc. Fusion proteins canbe prepared by fusing commercially available DNA encoding these peptidesor proteins with a DNA encoding a protein of the present invention, andexpressing the fused DNA thus prepared.

In addition, as a method that is well known to those skilled in the artfor preparing proteins that are functionally equivalent to a knownprotein, hybridization techniques can be employed (Sambrook J. et al.(1989) Molecular Cloning 2nd ed., 9.47-9.58, Cold Spring Harbor Lab.press). More specifically, those skilled in the art may readily isolateDNAs having high homology to the DNA sequences (SEQ ID NO: 1 or 3)encoding the 285 proteins, based on the entire DNA sequence or partsthereof, and isolate DNA-encoding proteins functionally equivalent tothe 285 proteins from these DNAs.

The present invention includes proteins that are functionally equivalentto a 285 protein, and which are encoded by DNAs that hybridizes with aDNA-encoding a 285 protein. Such proteins include, for example,homologues from mice and other mammals (e.g., proteins from human, rats,rabbits, cattle, and so on).

Hybridization conditions for isolating DNAs encoding proteins that arefunctionally equivalent to a 285 protein can be appropriately selectedby those skilled in the art. Conditions for hybridization, for example,may be those with low stringency. Low stringency conditions means thatthe washing conditions after hybridization are, for example, 42° C.,0.1×SSC, and 0.1% SDS, or preferably 50° C., 0.1×SSC, and 0.1% SDS.Examples of hybridization conditions that are more preferable areconditions with high stringency. An example of high stringencyconditions is 65° C., 5×SSC and 0.1% SDS. Under these conditions, thehigher the temperature, the higher the homology of the obtained DNA.However, several factors such as temperature and salt concentration caninfluence the stringency of hybridization and one skilled in the art canappropriately select these factors to achieve a similar stringency.

However, instead of hybridization, DNA encoding functionally equivalentproteins to a 285 protein can be isolated via gene amplification methodslike polymerase chain reaction (PCR), which uses primers that aresynthesized based on the sequence information of a DNA encoding a 285protein (SEQ ID NO: 1 or 3).

A protein that is functionally equivalent to a 285 protein, encoded by aDNA that is isolated by such hybridization techniques and geneamplification techniques, will normally have a high amino acid sequencehomology to a 285 protein (SEQ ID NO: 2 or 4). The proteins of thisinvention also include proteins that are functionally equivalent to a285 protein in addition to having a high sequence homology to the aminoacid sequence of the 285 protein. High sequence homology of a proteintypically means that in its amino acids, usually a homology of at least50% or more is present, preferably a homology of 75% or more, morepreferably a homology of 85% or more, and most preferably a homology of95% or more. In order to determine the homology of a protein, analgorithm that is described in the literature can be used (Wilbur W J.and Lipman D J. (1983) Proc. Natl. Acad. Sci. USA 80, 726-730).

The proteins of this invention may have different amino acid sequences,molecular weights, and isoelectric points. They may also havedifferences in the presence or absence of sugar chains and their forms,depending on the cells or hosts that produce these proteins or theproduction method (described later). However, so long as the obtainedprotein has the same function as a 285 protein, it is within the scopein the present invention. For example, if a protein of this invention isexpressed in a prokaryotic cell such as E. coli a methionine residuewill be added to the N terminus of the amino acid sequence of theoriginal protein. The proteins of this invention will also include suchproteins.

The proteins of the present invention can be prepared as recombinantproteins or naturally occurring proteins, via methods well known bythose skilled in the art. A recombinant DNA can be prepared by insertinga DNA (for example, the DNA comprising the nucleotide sequence of SEQ IDNOs: 1 or 3) which encodes a protein of the present invention into anappropriate vector, collecting the recombinant obtained by introducingthe vector into appropriate host cells, obtaining the extract, andpurifying by subjecting the extract to chromatography such as ionexchange, reverse, gel filtration, or affinity chromatography in whichan antibody against a protein of the present invention is fixed oncolumn or by combining more than one of these columns.

In addition, when a protein of the present invention is expressed inhost cells (for example, animal cells and E. coli) as a fusion proteinwith glutathione-S-transferase protein or as a recombinant proteinsupplemented with multiple histidines, the expressed recombinant proteincan be purified using a glutathione column or a nickel column. Afterpurifying the fusion protein, it is also possible to exclude regionsother than the objective protein by cutting with thrombin or factor-Xa,as required.

A naturally occurring protein can be isolated by methods known by aperson skilled in the art, for example, using an affinity column inwhich the antibody binding to a protein of the present invention(described below) is bound against an extract of tissues or cellsexpressing a protein of the present invention. The antibody can be apolyclonal or a monoclonal antibody.

The present invention also contains partial peptides of the proteins ofthe present invention. A partial peptide of the present inventioncomprises at least 7 amino acids or more, preferably 8 amino acids ormore, and more preferably 9 amino acids or more. The partial peptidescan be used, for example, for preparing an antibody against a protein ofthe present invention, screening a compound binding to a protein of thepresent invention, and for screening accelerators or inhibitors of aprotein of the present invention. The partial peptides can also be usedas antagonists or competitive inhibitors against a protein of thepresent invention. Because the 285 proteins of the present inventionhave a GC-box binding activity, nuclear localization activity andtranscription regulating activity, partial peptides of this inventioninclude partial peptides having at least one activity among theseactivities. A partial peptide of the invention can be produced bygenetic engineering, known methods of peptide synthesis, or by digestinga protein of the invention with an appropriate peptidase. For peptidesynthesis, for example, solid phase synthesis or liquid phase synthesismay be employed.

A DNA encoding a protein of the present invention can be used for theproduction of the protein in vivo or in vitro as described above. A DNAencoding a protein of the present invention can also be used forapplication to gene therapy for diseases attributed to geneticabnormality in the gene encoding a protein of the present invention. Anyform of the DNA can be used, as long as it encodes a protein of thepresent invention. Specifically, cDNA synthesized from mRNA, genomicDNA, or chemically synthesized DNA can be used. The present inventionincludes a DNA comprising a given nucleotide sequence based ondegeneracy of genetic codons, as long as it encodes a protein of thepresent invention.

A DNA of the present invention can be prepared by methods known to thoseskilled in the art. For example, a DNA of the present invention can beprepared from a cDNA library from cells which express a protein of thepresent invention by conducting hybridization using a partial sequenceof a DNA of the present invention (e.g., SEQ ID NO: 1 or 3) as a probe.A cDNA library can be prepared, for example, by the method described bySambrook J. et al. (Sambrook J. et al. (1989) Molecular Cloning, ColdSpring Harbor Laboratory Press), or by using commercially available cDNAlibraries. A cDNA library can be also prepared by extracting RNA fromcells expressing a protein of the present invention, synthesizing cDNAusing reverse transcriptase, synthesizing an oligo DNA base on thesequence of the DNA of the present invention (for example, SEQ ID NOs: 1or 3), conducting PCR using these as primers, and amplifying cDNAencoding the protein of the present invention.

In addition, by sequencing the nucleotides of the obtained cDNA, atranslation region encoded by the cDNA can be determined, and the aminoacid sequence of a protein of the present invention can be obtained.Moreover, by screening the genomic DNA library using the obtained cDNAas a probe genomic DNA can be isolated.

More specifically, mRNAs may first be prepared from a cell, tissue, ororgan (for example, testis, brain, heart, liver, and kidney) in which aprotein of the invention is expressed. Known methods can be used toisolate mRNAs. For instance, total RNA is prepared by the guanidineultracentrifugation method (Chirgwin J M. et al. (1979) Biochemistry 18,5294-5299) or by the AGPC method (Chomczynski P. and Sacchi N. (1987)Anal. Biochem. 162, 156-159), and mRNA is purified from total RNA usingmRNA Purification Kit (Pharmacia). Alternatively, mRNA may be directlypurified by the QuickPrep mRNA Purification Kit (Pharmacia).

The obtained mRNA is used to synthesize cDNA using reversetranscriptase. A cDNA may be synthesized using kits, such as the AMVReverse Transcriptase First-strand cDNA Synthesis Kit (Seikagaku Kogyo).Alternatively, a cDNA may be synthesized and amplified according to the5′-RACE method (Frohman M A. et al. (1988) Proc. Natl. Acad. Sci. U.S.A.85, 8998-9002; Belyavsky A. et al. (1989) Nucleic Acids Res.17:2919-2932) wherein primers, described herein, the 5′-Ampli FINDERRACE Kit (Clontech), and polymerase chain reaction (PCR) are used.

A desired DNA fragment is prepared from the PCR products and ligatedwith a vector DNA. The recombinant vectors are used to transform E. coliand a desired recombinant vector is prepared from a selected colony. Thenucleotide sequence of the desired DNA can be verified by conventionalmethods, such as, the dideoxynucleotide chain termination method.

A DNA of the invention may be also designed to have a sequence that isexpressed more efficiently by taking into account the frequency of codonusage in the host to be used for expression (Grantham R. et al. (1981)Nucleic Acids Res. 9, 43-74). A DNA of the present invention may bealtered by a commercially available kit or a conventional method. Forinstance, a DNA may be altered by digestion with restriction enzymes,insertion of synthetic oligonucleotides or appropriate DNA fragments,addition of linkers, or insertion of the initiation codon (ATG) and/orthe stop codon (TAA, TGA, or TAG) The DNAs of the present inventionspecifically encompass a DNA consisting of the nucleotide sequence fromthe 75^(th) nucleotide Adenine to the 1271^(st) nucleotide Adenine ofSEQ ID NO: 1, and a DNA comprising this nucleotide sequence. Further, aDNA consisting of the nucleotide sequence from the 1^(st)nucleotideAdenine to the 1197^(th) nucleotide Adenine of SEQ ID NO: 3,and a DNA comprising this nucleotide sequence are also encompassed bythe DNAs of the present invention.

The DNAs of this invention include a DNA that (a) hybridizes with a DNAconsisting of the nucleotide sequence of SEQ ID NO: 1 or 3; and (b)encodes a protein that is functionally equivalent to a protein of thisinvention mentioned above. Conditions for hybridization can be selectedappropriately by those skilled in the art, and those conditionsspecifically mentioned above may be used. Under these conditions, DNAhaving higher homology is obtained as the temperature is raised. Theabove-mentioned DNA to be hybridized is preferably a naturally occurringDNA, for example, a cDNA or chromosomal DNA.

The present invention also provides vectors into which a DNA of thepresent invention is inserted. The vectors of the present invention areuseful to retain a DNA of the present invention in host cell, or toexpress a protein of the present invention.

When E. coli is used as the host cell and a vector is amplified thereinto produce a large amount in E. coli (e.g., JM109, DH5α, HB101, orXL1Blue), the vector should have an “ori” that may be amplified in E.coli and a marker gene (e.g., ampicillin, tetracycline, kanamycin, orchloramphenicol)) that enables selection of transformed E. coli (e.g., adrug-resistance gene selected by a drug. For example, the M13-seriesvectors, the pUC-series vectors, pBR322, pBluescript, pCR-Script, etc.can be used. In addition to the vectors described above, pGEM-T,pDIRECT, and pT7 can also be used for subcloning and extracting cDNA.When a vector is used to produce a protein of the present invention, anexpression vector is especially useful. For example, an expressionvector to be expressed in E. coli should have the above characteristicsto be amplified in E. coli. When E. coli (e.g., JM109, DH5α, HB101 orXL1 Blue) are used as the host cell, in addition to the abovecharacteristics, the vector should have a promoter like the laczpromoter (Ward et al. (1989) Nature 341, 544-546; (1992) FASEB J. 6,2422-2427), the araB promoter (Better et al. (1988) Science 240,1041-1043), or the T7 promoter that can efficiently express the desiredgene in E. coli. As such a vector, for example, pGFX-5X-1 (Pharmacia),“QIAexpress system” (Qiagen), pEGFP or pET (in this case, the host ispreferably BL21 which expresses T7 RNA polymerase) can be used inaddition to the above vectors.

A vector also may contain a signal sequence for polypeptide secretion.As a signal sequence for protein secretion, the pelB signal sequence(Lei S P. et al. (1987) J. Bacteriol. 169, 4379) can be used in the caseof producing proteins into the periplasm of E. coli. For introducing avector into host cells, for example, the calcium chloride method and theelectroporation method can be used.

Besides E. coli, expression vectors derived from mammals (for example,pcDNA3 (Invitrogen) and pEGF-BOS (Nucleic Acids. Res. 1990, 18 (17),p5322), pEF, pCDM8); expression vectors derived from insect cells (forexample, “Bac-to-BAC baculovirus expression system” (GIBCO BRL),pBacPAK8); expression vectors derived from plants (for example pMH1,pMH2); expression vectors derived from animal viruses (for example,pHSV, pMV, pAdexLcw); expression vectors derived from retroviruses (forexample, pZIPneo); expression vectors derived from yeast (for example,“Pichia Expression Kit” (Invitrogen), pNV11, SP-Q01); expression vectorsderived from Bacillus subtilis (for example, pPL608, pKTH50) can be usedas vectors for producing a protein of the present invention.

In order to express a vector in animal cells, such as CHO, COS, orNIH3T3 cells, the vector should have a promoter necessary for expressionin such cells, for example, the SV40 promoter (Mulligan et al. (1979)Nature 277, 108), the MMLV-LTR promoter, the EF1α promotor (Mizushima etal. (1990) Nucleic Acids Res. 18, 5322), or the CMV promoter, etc. andpreferably a marker gene for selecting transformants (for example, adrug resistance gene selected by a drug (e.g., neomycin, G418)).Examples of vectors with these characteristics include, but are notlimited to, pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, pOp13, and so on.

In addition, methods to stably express a gene and amplifying the copynumber of the gene in cells include: for example, a method wherein avector comprising the complementary DHFR gene (for example pCHO I) isintroduced into CHO cells with deleted nucleic acid synthesizingpathway, and the vector is amplified by the addition of methotrexate(MTX). On the other hand, in the case of transient expression of a gene,a method wherein a vector (e.g., pcD) comprising replication origin ofSV40 is transformed using COS cells comprising the SV40 T antigenexpressing gene on chromosomes can be used. The origin used forreplication may be those of polyomavirus, adenovirus, bovine papillomavirus (BPV),etc. In addition, the expression vector may include aselection marker gene for amplification of the gene copies in hostcells. Examples of such markers include, but are not limited to, theaminoglycoside transferase (APH) gene, the thymidine kinase (TK) gene,the E. coli xanthine-guanine phosphoribosyl transferase (Ecogpt) gene,and the dihydrofolate reductase (dhfr) gene.

On the other hand, a gene of the present invention can be expressed invivo in animals, for example, by inserting a DNA of the presentinvention into an appropriate vector and introducing the vector in vivoby conventional methods, such as the retrovirus method, the liposomemethod, the cationic liposome method, and the adenovirus method.According to such methods, gene therapy against diseases attributed tomutation of the h285 gene of the present invention can be affected. As avector, for example, adenovirus vector (for example pAdexlcw), andretrovirus vector (for example, pZIPneo) can be used; however, thepresent invention is not restricted thereto. Common gene manipulation,for example, insertion of a DNA of the present invention into a vector,can be performed according to any standard method (Molecular Cloning,5.61-5.63) Administration into a living body can be either an ex vivomethod, or in vivo method.

The present invention provides a transformed cell that retains a DNA orvector of the present invention. The host cell into which a vector ofthe invention is introduced is not particularly limited. E. coli orvarious animal cells can be used. The transformed cells of the presentinvention can be used as a production system for producing or expressinga protein of the present invention. The present invention providesmethods of producing a protein of the invention either in vitro or invivo. For in vitro production, eukaryotic cells or prokaryotic cells canbe used as host cells.

Useful eukaryotic cells as host include animal, plant, or fungi cells.The following can be used: animal cells/mammalian cells, such as CHO (J.Exp. Med. 108, 945 (1995)), COS, 3T3, myeloma, baby hamster kidney(BHK), HeLa, and Vero cells; amphibian cells, such as Xenopus oocytes(Valle et al. (1981) Nature 291,340-358); or insect cells, such as Sf9,Sf21, and Tn5 cells. CHO cells lacking the DHFR gene (dhfr-CHO) (Proc.Natl. Acad. Sci. U.S.A. 77, 4216-4220 (1980)) or CHO K-1 (Proc. Natl.Acad. Sci. U.S.A. 60, 1275 (1968)) may also be used. In animal cells,CHO cells are particularly preferable for mass expression. A vector canbe introduced into host cells by, for example via the calcium phosphatemethod, the DEAE dextran method, the cationic liposome DOTAP (BoehringerMannheim), the electroporation method, or the lipofection method.

Plant cells originating from Nicotiana tabacum are a knownprotein-production system and may be used as callus cultures. Fungicells, yeast cells such as Saccharomyces (including Saccharomycescerevisiae) or filamentous fungi, such as, Aspergillus (includingAspergillus niger) are known and may be used herein.

Useful prokaryotic cells include bacterial cells, such as, E. coli, forexample, JM109, DH5α, HB101 are known. Regarding others, Bacillussubtilis is known.

These host cells are transformed by a desired DNA, and the resultingtransformants are cultured in vitro to obtain a protein. Transformantscan be cultured using known methods. Culture medium for animal cell, forexample, DMEM, MEM, RPMI1640, or IMDM may be used with or without serumsupplement such as fetal calf serum (FCS). The pH of the culture mediumis preferably between about pH 6 to 8. Such cells are typically culturedat about 30 to 40° C. for about 15 to 200 hr, and the culture medium maybe replaced, aerated, or stirred if necessary.

Animal and plant hosts may be used for in vivo production. For example,a desired DNA can be introduced into an animal or plant host. Encodedproteins are produced in vivo, and then recovered. These animal andplant hosts are included in the host cells of the present invention.

Animals to be used for the production systems described above include,but are not limited to, mammals and insects. Mammals, such as, goat,porcine, sheep, mouse and bovine may be used (Vicki Glaser (1993)SPECTRUM Biotechnology Applications). Alternatively, the mammals may betransgenic animals.

For instance, a desired DNA may be prepared as a fusion gene with a geneencoding a protein specifically produced in milk, such as goat β casein.DNA fragments comprising a fusion gene having the desired DNA areinjected into goat embryos, which are then introduced back to femalegoats. Proteins are recovered from milk produced by the transgenic goats(i.e., those born from the goats that had received the modified embryos)or from their offspring. To increase the amount of milk containing theproteins produced by transgenic goats, appropriate hormones may beadministered to them (Ebert K M. et al. (1994) Bio/Technology 12,699-702).

Alternatively, insects like the silkworm may be used. A desired DNAinserted into baculovirus can be used to infect silkworms, and a desiredprotein is then recovered from their body fluid (Susumu M. et al. (1985)Nature 315, 592-594).

As plants, tobacco can be used. In use of tobacco, a desired DNA isinserted into a plant expression vector, such as pMON530, which is thenintroduced into bacteria, such as Agrobacterium tumefaciens. Then, thebacteria are used to infect tobacco like Nicotiana tabacum, and adesired polypeptide is recovered from the leaves of the plant (Julian K.-C. Ma et al. (1994) Eur. J. Immunol. 24, 131-138).

A protein of the present invention obtained as above may be isolatedfrom the interior or exterior (e.g., medium) of the cells or hosts, andpurified as a substantially pure homogeneous protein. The method forprotein isolation and purification is not limited to any specificmethod. In fact, any standard method may be used. For instance, columnchromatography, filtration, ultrafiltration, salt precipitation, solventprecipitation, solvent extraction, distillation, immunoprecipitation,SDS-polyacrylamide gel electrophoresis, isoelectric pointelectrophoresis, dialysis, and recrystallization may be appropriatelyselected and combined to isolate and purify the protein.

For chromatography, for example, affinity chromatography, ion-exchangechromatography, hydrophobic chromatography, gel filtration, reversephase chromatography, adsorption chromatography, and such may be used(ed. Daniel R. Marshak et al. (1996) Strategies for Protein Purificationand Characterization: A Laboratory Course Manual., Cold Spring HarborLaboratory Press). These chromatographies may be performed by liquidchromatography, such as, HPLC and FPLC. Thus, the present inventionprovides highly purified proteins produced by the above methods.

A protein of the present invention may be optionally modified orpartially deleted by treating it with an appropriateprotein-modification enzyme before or after purification. Usefulprotein-modification enzymes include, but are not limited to, trypsin,chymotrypsin, lysylendopeptidase, protein kinase, and glucosidase.

The present invention provides antibodies that bind to a protein of theinvention. An antibody of the invention can be used in any form, such asmonoclonal or polyclonal antibodies, and includes antiserum obtained byimmunizing a rabbit with a protein of the invention, all classes ofpolyclonal and monoclonal antibodies, human antibodies, and humanizedantibodies produced via genetic recombination.

A protein of the invention used as an antigen to obtain an antibody maybe derived from any animal species, but is preferably derived from amammal such as a human, mouse, or rat, more preferably from a human. Ahuman-derived protein may be obtained from the nucleotide or amino acidsequences disclosed herein.

In the present invention, a protein to be used as an immunizationantigen may be a complete protein or a partial peptide of a protein. Apartial peptide may be, for example, an amino (N)-terminal or carboxy(C)-terminal fragment of the protein. Herein, “an antibody” is definedas an antibody that specifically reacts with either the full-length or afragment of a protein.

A gene encoding a protein of the invention or its fragment may beinserted into a known expression vector, which is then used to transforma host cell as described herein. The desired protein or its fragment maybe recovered from the exterior or interior of the host cells by anystandard method, and may be used as an antigen. Alternatively, cellsexpressing the protein or their lysates, or a chemically synthesizedprotein may be used as an antigen. Short peptides are preferably boundwith carrier proteins such as bovine serum albumin, ovalbumin, andkeyhole limpet hemocyanin to be used as the antigen.

Any mammalian animal may be immunized with the antigen, but preferablythe compatibility with parental cells used for cell fusion is taken intoaccount.

In general, animals of the orders Rodentia, Lagomorpha, or Primate areused. Animals of Rodentia, rodents, include, for example, mouse, rat,and hamster. Animals of Lagomorpha, lagomorphs, include, for example,rabbit. Animals of Primate, primates, include, for example, a monkey ofcatarrhine (old world monkey) such as Macaca fascicularis, rhesusmonkey, sacred baboon, or chimpanzee.

Methods for immunizing animals against antigens are known in the art.Intraperitoneal injection or subcutaneous injection of antigens is usedas a standard method for immunization of mammals. More specifically,antigens may be diluted and suspended in an appropriate amount withphosphate buffered saline (PBS),physiological saline, etc. If desired,the antigen suspension may be mixed with an appropriate amount of astandard adjuvant, such as Freund's complete adjuvant, made intoemulsion, and then administered to the mammalian animals. Preferably, itis followed by several administrations of antigen mixed with anappropriately amount of Freund's incomplete adjuvant every 4 to 21 days.An appropriate carrier may also be used for immunization. Afterimmunization as above, serum is examined for increase of the amount ofdesired antibodies by a standard method.

Polyclonal antibodies against a protein of the present invention may beprepared by collecting blood from the immunized mammal examined for theincrease of desired antibodies in the serum, and separating serum fromthe blood by any conventional method. Polyclonal antibodies may be usedas serum containing the polyclonal antibodies, or if necessary, afraction containing the polyclonal antibodies may be isolated from theserum. Immunoglobulin G or M can be prepared by obtaining a fractionwhich recognizes only a protein of the present invention using anaffinity column coupled with the protein of the present invention andfurther purifying this fraction using protein A or protein G column.

To prepare monoclonal antibodies, immune cells are collected from themammal immunized against an antigen and checked for the increased levelof desired antibodies in the serum as described above, and are subjectedto cell fusion. It is preferred that the immune cells used for cellfusion be obtained from spleen. Other parental cells can be fused withthe above immunocyte. For example, preferably myeloma cells ofmammalians and more preferably myeloma cells that acquired the propertyfor selecting fused cells by drugs, can be used.

The above immunocyte and myeloma cells can be fused by known methods,for example, the method by Milstein et al. (Galfre G. and Milstein C.(1981) Methods Enzymol. 73, 3-46).

Resulting hybridomas obtained by the cell fusion may be selected bycultivating them in a standard selection medium, such as the HAT medium(medium containing hypoxanthine, aminopterin, and thymidine) The cellculture is typically continued in the HAT medium for several days toseveral weeks, a sufficient time to allow all the other cells, exceptdesired hybridoma (non-fused cells), to die. Then, by the standardlimiting dilution method, a hybridoma cell producing the desiredantibody is screened and cloned.

In addition to the above method, in which a non human animal isimmunized against an antigen for preparing hybridoma, human lymphocytes,such as that infected by EB virus, may be immunized with a protein,protein expressing cells, or their lysates in vitro. Then, the immunizedlymphocytes are fused with human-derived myeloma cells capable ofindefinitely dividing, such as U266, to yield a hybridoma producing adesired human antibody having binding ability to the protein (UnexaminedPublished Japanese Patent Application (JP-A) No. Sho 63-17688).

Next, the monoclonal antibody, obtained by transplanting the obtainedhybridomas into the abdominal cavity of a mouse and by extractingascites, can be purified by, for example, ammonium sulfateprecipitation, protein A or protein G column, DEAE ion exchangechromatography, or an affinity column to which a protein of the presentinvention is coupled. An antibody of the present invention can be usedfor not only purification and detection of a protein of the presentinvention, but also as a candidate for agonists and antagonists of aprotein of the present invention. In addition, an antibody can beapplied to antibody treatment for diseases associated with a protein ofthe present invention. When the obtained antibody is used for theadministration to the human body (antibody treatment), a human antibodyor a humanized antibody is preferable for reducing immunogenicity.

For example, transgenic animals having a repertory of human antibodygenes may be immunized against a protein, protein expressing cells, ortheir lysates as an antigen. Antibody producing cells are collected fromthe animals and fused with myeloma cells to obtain hybridomas, fromwhich human antibodies against a protein can be prepared (seeWO92-03918, WO93-2227, WO94-02602, WO94-25585, WO96-33735, andWO96-34096).

Alternatively, an immune cell, such as an immunized lymphocyte,producing antibodies may be immortalized by an oncogene and used forpreparing monoclonal antibodies.

Monoclonal antibodies thus obtained can be also recombinantly preparedusing genetic engineering techniques (see, Borrebaeck C A K. and LarrickJ W. (1990) THERAPEUTIC MONOCLONAL ANTIBODIES, published in the UnitedKingdom by MACMILLAN PUBLISHERS LTD). A DNA encoding an antibody may becloned from an immune cell, such as a hybridoma or an immunizedlymphocyte producing the antibody, inserted into an appropriate vector,and introduced into host cells to prepare a recombinant antibody. Thepresent invention also provides recombinant antibodies prepared asdescribed above.

Furthermore, an antibody of the present invention may be a fragment ofan antibody or modified antibody, as long as it binds to one or more ofthe proteins of the present invention. For instance, the antibodyfragment may be Fab, F(ab′)₂, Fv, or single chain Fv (scFv) in which Fvfragments from H and L chains are ligated by an appropriate linker(Huston J S. et al. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5879-5883).More specifically, an antibody fragment may be generated by treating anantibody with enzymes like papain or pepsin. Alternatively, a geneencoding an antibody fragment may be constructed, inserted into anexpression vector, and expressed in an appropriate host cell (see, forexample, Co MS. et al. (1994) J. Immunol. 152, 2968-2976; Better M. andHorwitz A H. (1989) Methods Enzymol. 178, 476-496; Pluckthun A. andSkerra A. (1989) Methods Enzymol. 178, 497-515; Lamoyi E. (1986) MethodsEnzymol. 121, 652-663; Rousseaux J. et al. (1986) Methods Enzymol. 121,663-669; Bird RE. and Walker BW. (1991) Trends Biotechnol. 9, 132-137).

An antibody may be modified by conjugation with a variety of molecules,such as polyethylene glycol (PEG). The present invention provides suchmodified antibodies. The modified antibody can be obtained by chemicallymodifying an antibody. These modification methods are conventional inthis field.

Alternatively, an antibody of the present invention may be obtained as achimeric antibody, where a variable region is derived from nonhumanantibody and the constant region is derived from human antibody; or as ahumanized antibody comprising the complementarity determining region(CDR) derived from nonhuman antibody, the frame work region (FR) derivedfrom human antibody, and the constant region.

Obtained antibodies may be purified into homogeneity. An antibody usedin the present invention can be separated and purified by conventionalmethods used for separating and purifying proteins. For example, theseparation and purification of a protein can be performed by anappropriately selected and combined use of column chromatography (suchas affinity chromatography), filter, ultrafiltration, salting-out,dialysis, SDS polyacrylamide gel electrophoresis, isoelectric focusing,etc. (Antibodies: A Laboratory Manual. Ed Harlow and David Lane, ColdSpring Harbor Laboratory, 1988). However, the present invention is notlimited to the above-mentioned techniques. The concentration ofantibodies obtained above can be determined by measuring absorbance, bythe enzyme-linked immunosorbent assay (ELISA), etc.

Examples of columns used for affinity chromatography include protein Acolumns and protein G columns. Examples of columns using protein Acolumn include Hyper D, POROS, Sepharose F. F. (Pharmacia) etc.

In addition to affinity chromatography, the chromatography includes, forexample, ion-exchange chromatography, hydrophobic chromatography, gelfiltration, reverse-phase chromatography, adsorption chromatography,etc. (Strategies for Protein Purification and Characterization: ALaboratory Course Manual. Ed Daniel R. Marshak et al., Cold SpringHarbor Laboratory Press, 1996). The chromatographic procedures can becarried out by liquid-phase chromatography such as HPLC, FPLC, or thelike.

For example, measurement of absorbance, enzyme-linked immunosorbentassay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), and/orimmunofluorescence may be used to measure the antigen binding activityof an antibody of the invention. In ELISA, an antibody of the presentinvention is immobilized on a plate, a protein of the invention isapplied to the plate, and then a sample containing a desired antibody,such as culture supernatant of antibody producing cells or purifiedantibodies, is applied. Then, a secondary antibody that recognizes theprimary antibody and is labeled with an enzyme, such as alkalinephosphatase, is applied, and the plate is incubated. Next, afterwashing, an enzyme substrate, such as p-nitrophenyl phosphate, is addedto the plate, and the absorbance is measured to evaluate the antigenbinding activity of the sample. A fragment of a protein, such as aC-terminal fragment, may be used as a protein. BIAcore (Pharmacia) maybe used to evaluate the activity of an antibody according to the presentinvention.

The above methods allow for the detection or measurement of the proteinsof the invention, by exposing an antibody of the invention to a sampleassumed to contain a protein of the invention, and detecting ormeasuring the immune complex formed by the antibody and the protein.Because the method of detection or measurement of proteins according tothe invention can specifically detect or measure proteins, the methodmay be useful in a variety of experiments in which the protein is used.

The present invention provides a polynucleotide having at least 15nucleotides that is complementary to a DNA that encodes a 285 protein(SEQ ID NO: 1 or 3) or the complementary strand thereof.

The term “complementary strand,” as employed herein, is defined as onestrand of a double strand DNA composed of A:T (in the case of RNA, A:U)and G:C base pairs to the other strand. In addition, “complementary” isdefined as not only those completely matching within a continuous regionof at least 15 nucleotides, but also having a homology of at least 70%,preferably at least 80%, more preferably 90%, and most preferably 95% orhigher within that region. The homology may be determined using thealgorithm described herein.

Probes or primers for detection and amplification of a DNA encoding aprotein of this invention or for detection of an expression of theprotein, or nucleotides or nucleotide derivatives for regulating proteinexpression (for example, antisense oligonucleotides and ribozymes, orDNA encoding them) are included in these polynucleotides. In addition,such polynucleotides may be also used for preparing DNA chips.

When used as a primer, the region on the 3′ side is designed to becomplementary to a DNA encoding a protein of the invention, andrestriction enzyme recognition sequence and tags can be added to the 5′side.

For example, an antisense oligonucleotide that hybridizes with a portionof the nucleotide sequence of SEQ ID NO: 1 or 3 is also included in theantisense oligonucleotides of the present invention. An antisenseoligonucleotide is preferably one against at least 15 continuousnucleotides in the nucleotide sequence of SEQ ID NO: 1 or 3. Morepreferably, it is an antisense oligonucleotide having at least 15continuous nucleotides that contains the translation initiation codon.

Derivatives or modified products of antisense oligonucleotides can beused as antisense oligonucleotides. Examples of such modified productsare, lower alkyl phosphonate modifications such asmethyl-phosphonate-type or ethyl-phosphonate-type, phosphothioatemodifications and phosphoamidate modifications.

The term “antisense oligonucleotides” as used herein, means not onlythose in which the entire nucleotides corresponding to thoseconstituting a specified region of a DNA or mRNA are complementary, butalso those having a mismatch of one or more nucleotides, so long as theDNA or mRNA and the oligonucleotide can hybridize to each other in anucleotide sequence (i.e., SEQ ID NO: 1 or 3) specific manner.

An antisense oligonucleotide derivative of the present invention hasinhibitory effect on the function of a protein of the present inventionas a result that the derivative inhibits the expression of the proteinof the invention by acting upon cells producing the protein of theinvention and by binding to the DNA or mRNA encoding the protein toinhibit its transcription or translation or to promote the degradationof the mRNA.

An antisense oligonucleotide derivative of the present invention can bemade into an external preparation, such as a liniment and a poultice, bymixing with a suitable base material which is inactive against thederivatives.

In addition, as necessary, the derivatives can be formulated intotablets, powders, granules, capsules, liposome capsules, injections,solutions, nose-drops, and freeze-drying agents and such by addingexcipients, isotonic agents, solubilizing agents, stabilizers,preservative substance, painkillers, etc. These can be prepared byfollowing usual methods.

An antisense oligonucleotide derivative of the present invention isgiven to a patient by directly applying onto the ailing site or byinjecting into a blood vessel so that it will reach the site of ailment.An antisense-mounting medium can also be used to increase durability andmembrane-permeability. Examples are liposome, poly-L-lysine, lipid,cholesterol, lipofectin or derivatives of these.

The dosage of an antisense oligonucleotide derivative of the presentinvention can be adjusted suitably according to the patient's conditionand used in desired amounts. For example, a dose range of 0.1 to 100mg/kg, preferably 0.1 to 50 mg/kg can be administered.

An antisense oligonucleotide of the invention inhibits the expression ofa protein of the invention and thereby is useful for suppressing thebiological activity of the protein of the invention. In addition,expression-inhibitors comprising an antisense oligonucleotide of theinvention are useful in that they can inhibit the biological activity ofa protein of the invention. It is thought that it is possible to use anantisense oligonucleotides of this invention for suppressing biologicalactivities of a protein of the invention.

A protein of the invention may be used for screening compounds bindingto the protein. Specifically, a protein may be used in methods ofscreening for compounds comprising the steps of: (1) contacting aprotein of the present invention to a test sample in which a compoundbinding to the protein is expected to be contained; and (2) selectingthe compound having the binding activity to the protein.

A protein of the present invention to be used for screening may be arecombinant protein, a protein derived from the nature, or partialpeptide thereof. Alternatively, the protein may be in a form expressedon a cell surface or in a form of cell membrane fraction. Any testsample, for example, cell extracts, cell culture supernatant, productsof fermenting microorganism, extracts from marine organism, plantextracts, purified or crude proteins, peptides, non-peptide compounds,synthetic low molecular compounds and naturally-occurring compounds, canbe used. A protein of the present invention can be contacted with a testsample in the following forms: as a purified protein, a soluble protein,a form bound to a carrier, a fusion protein with another protein, a formexpressed on cell membrane, or a cell membrane fraction.

By using a protein of the present invention, for example, in a methodfor screening for proteins binding to the protein thereof, many methodswell known by a person skilled in the art can be used. Such a screeningcan be conducted by, for example, the immunoprecipitation method,specifically, in the following manner. A gene encoding a protein of thepresent invention is expressed in a host cell, such as an animal cell,by inserting the gene into an expression vector for foreign gene, suchas pSV2neo, pcDNA I, pCD8. As a promoter to be used for the expression,any promoter which can be generally used can be selected; for example,the SV40 early promoter (Rigby in Williamson (ed.) (1982) Geneticengineering, vol.3. Academic Press, London, p. 83-141), the EF-1αpromoter (Kim et al. (1990) Gene 91, 217-223), the CAG promoter (Niwa etal. (1991) Gene 108, 193-200) the RSV LTR promoter (Cullen (1987)Methods in Enzymology 152, 684-704), the SRα promoter (Takebe et al.(1988) Mol. Cell. Boil. 8, 466), the CMV immediate early promoter (Seedand Aruffo (1987) Proc. Natl. Acad. Sci USA 84, 3365-3369), the SV40late promoter (Gheysen and Fiers (1982) J. Mol. Appl. Genet. 1,385-394), the Adenovirus late promoter (Kaufman et al. (1989) Mol. Cell.Biol. 9, 946), the HSV TK promoter, and so on may be used.

To express a foreign gene by introducing the gene into animal cells, theelectroporation method (Chu G. et al. (1987) Nucl. Acid Res. 15,1311-1326), the calcium phosphate method (Chen C and Okayama H. (1987)Mol Cell. Biol. 7, 2745-2752), the DEAE dextran method (Lopata M A. etal. (1984) Nucl. Acids Res. 12, 5707-5717; Sussman D J. and Milman G.(1985) Mol. Cell. Biol. 4, 1642-1643), the Lipofectin method (DerijardB. (1994) Cell 7, 1025-1037; Lamb B T. et al. (1993) Nature Genetics 5,22-30; Rabindran S K. et al. (1993) Science 259, 230-234) etc. can beused.

A protein of the present invention can be expressed as a fusion proteincomprising a recognition site (epitope) of a monoclonal antibody byintroducing the epitope of the monoclonal antibody, whose property hasbeen revealed, to N or C terminus of the protein of the presentinvention. A commercially available epitope-antibody system can be used(Experimental medicine 13, 85-90 (1995)). Through a multiple cloningsite, a vector which can express a fusion protein with, for example,β-galactosidase, maltose-binding protein, glutathione S-transferase,green florescence protein (GFP), is available in the market.

Methods have been reported in which fusion proteins are prepared byintroducing only small epitopes comprising several to a dozen of aminoacids, so that the properties of the proteins of the present inventionmay not change by making the proteins fusion proteins. Epitopes, forexample, polyhistidine (His-tag), influenza aggregate HA, human c-myc,FLAG, Vesicular stomatitis virus glycoprotein (VSV-GP), T7 gene 10protein (T7-tag), human simple herpes virus glycoprotein (HSV-tag),epitope such as E-tag (an epitope on monoclonal phage), and monoclonalantibodies recognizing these can be used as an epitope-antibody systemfor screening a protein binding to a protein of the present invention(Experimental Medicine 13, 85-90 (1995)).

In the immunoprecipitation, an immune complex is formed by adding theseantibodies to cell-eluate prepared using an appropriate detergent. Thisimmune complex comprises a protein of the present invention, a proteinhaving a binding affinity for the protein, and an antibody.Immunoprecipitation can be conducted with an antibody against a proteinof the present invention, besides-using antibodies against the aboveepitopes. An antibody against a protein of the present invention can beprepared, for example, by introducing a gene encoding the protein of thepresent invention into an appropriate E. coli expression vector;expressing the gene in E. coli; purifying the expressed protein; andimmunizing animals, for example, rabbits, mice, rats, goats, domesticfowls, etc. with such protein. The antibody can be prepared also byimmunizing the above animals against a synthesized partial peptide of aprotein of the present invention.

An immune complex can be precipitated, for example, by Protein ASepharose or Protein G Sepharose when the antibody is mouse IgGantibody. When a protein of the present invention is prepared as afusion protein with an epitope, for example GST, an immune complex canbe formed by using a substance specifically binding to these epitopes,such as glutathione-Sepharose 4B, in the same manner as in the use of anantibody-against a protein of the present invention.

Popular Immunoprecipitation can be performed by following or accordingto, for example, the reference (Harlow, E. and Lane, D. (1988)Antibodies, Cold Spring Harbor Laboratory publications, New York, pp.511-552,).

SDS-PAGE is commonly used for analysis of immunoprecipitated proteinsand the binding protein can be analyzed depending on the molecularweight of the protein by using gel with an appropriate concentration. Ingeneral, because it is difficult to detect a protein binding to aprotein of the present invention by a common staining method likeCoomassie staining or silver staining, the detection sensitivity for theprotein can be improved by culturing in a culture medium containing theradioactive isomer, ³⁵S-methionineor ³⁵S-cystein, labeling proteins inthe cells, and detecting the proteins. The target protein can bepurified from the SDS-polyacrylamide gel and its sequence can bedetermined directly after the molecular weight of the protein isdetermined.

Moreover, to isolate proteins that bind to a protein of the presentinvention by using the protein, for example, West western blotting(Skolnik E Y. et al. (1991) Cell 65, 83-90) may be used. Morespecifically, it is conducted as follows: (1) constructing a cDNAlibrary using a phage vector (λgt11, ZAP, etc.) from cells, tissues, andorgans (for example, testis, brain, heart, liver, and kidney) that areexpected to express binding proteins that bind to the protein of thisinvention; (2) expressing the cDNA library on LB-agarose andimmobilizing the expressed protein onto a filter; (3) reacting thepurified and labeled protein of this invention with the filter; and (4)detecting the plaque expressing the protein that binds to the protein ofthis invention by the label. Methods to label a protein of thisinvention may be a method that utilizes the binding characteristics ofbiotin and avidin; a method utilizing antibodies that bind specificallyto the protein of this invention or to peptides or polypeptides fused tothe protein of this invention (for example GST and such); a method thatutilizes radioisotopes; a method that utilizes fluorescence; and such.

Further, another embodiment of the screening method of this invention isexemplified by a method utilizing the two-hybrid system using cells(Fields S. and Sternglanz R. (1994) Trends. Genet. 10, 286-292; DaltonS. and Treisman R. (1992) “Characterization of SAP-1, a proteinrecruited by serum response factor to the c-fos serum response element.”Cell 68, 597-612; “MATCHMAKER Two-Hybrid System”, “Mammalian MATCHMAKERTwo-Hybrid Assay Kit”, “MATCHMAKER One-Hybrid System” (all manufacturedby Clonetech); and “HybriZAP Two-Hybrid Vector System” (manufactured byStratagene)). In the two-hybrid system, a protein of this invention or apartial peptide thereof may be fused to the DNA binding region of SRF orGAL4, and expressed in yeast. A cDNA library is constructed from cellspredicted to express proteins that bind to the protein of thisinvention, wherein the cDNA library is constructed in such a way thatthe proteins are expressed as fusion proteins with transcriptionactivation regions of VP16 or GAL4. The cDNA library is transfected intothe above yeast, and then positive clones are detected to isolate thecDNA derived from the library (expression of a protein that binds to theprotein of the invention in yeast leads to the binding of the twoproteins, and results in the activation of the reporter gene, whichallows detecting positive clones) The protein encoded by the isolatedcDNA may be obtained by introducing the cDNA into E. coli and expressingit therein. Thus, it is possible to prepare proteins that bind to aprotein of this invention and genes encoding them. The reporter geneused in the two-hybrid system may be such as Ade2 gene, Lac Z gene, CATgene, luciferase gene, PAI-1 (Plasminogen activator inhibitor type 1)gene, and such besides HIS3 gene, but are not limited to these examples.Screening by the two-hybrid method can also be performed using, inaddition to yeast, mammalian cells, etc.

A protein binding to a protein of the present invention can be screenedusing affinity chromatography. For example, a preferred method forscreening of the present invention utilizes affinity chromatography. Aprotein of the invention is immobilized on a carrier of an affinitycolumn, and a test sample, in which a protein capable of binding to theprotein of the invention is supposed to be expressed, is applied to thecolumn. A test sample herein may be, for example, cell extracts, celllysates, etc. After loading the test sample, the column is washed, andproteins bound to the protein of the invention can be prepared.

The amino acid sequence of the obtained protein is analyzed, an oligoDNA is synthesized based on the sequence, and cDNA libraries arescreened using the DNA as a probe to obtain a DNA encoding the protein.

A biosensor using the Surface Plasmon Resonance phenomenon may be usedas a means for detecting or quantifying the bound compound in thepresent invention. When such a biosensor is used, the interactionbetween a protein of the invention and a test compound can be observedin real-time as a surface plasmon resonance signal, using only a minuteamount of proteins without labeling (for example, BIAcore, Pharmacia).Therefore, it is possible to evaluate the binding between a protein ofthe invention and a test compound using a biosensor such as BIAcore.

Methods of screening molecules that bind when an immobilized protein ofthe present invention is exposed to synthetic chemical compounds,natural substance banks, or a random phage peptide display library, andmethods of screening using high-throughput based on combinatorialchemistry techniques (Wrighton N C., Farrel F X., Chang R., Kashyap AK., Barbone F P., Mulcahy L S., Johnson D L., Barret R W., Jolliffe LK., and Dower W J. (Jul. 26, 1996) “Small peptides as potent mimetics ofthe protein hormone erythropoietin” Science (UNITED STATES) 273, 458-64;Verdine G L. (Nov. 7, 1996) “The combinatorial chemistry of nature.”Nature (ENGLAND) 384, 11-13; Hogan J C Jr. (Nov. 7, 1996) “Directedcombinatorial chemistry.” Nature (ENGLAND) 384, 17-9) are well known tothose skilled in the art as methods for isolating not only proteins butalso chemical compounds that bind to a protein of the present invention(including agonist and antagonist).

A protein of the present invention is also useful for the screening forcompounds that control the transcriptional regulation activity of theprotein. More specifically, the protein of the present invention is usedin the method of screening for compounds that control thetranscriptional regulation activity of the protein of the presentinvention comprising the steps of: (a) contacting the protein of thepresent invention or partial peptides thereof with a test sampleexpected to contain a compound that controls the transcriptionalregulation activity of the protein; (b) detecting the transcriptionalregulation activity of the protein or partial peptides thereof; and (c)selecting the compound that decreases or increases the activity ascompared with that observed in the absence of the test sample (control).

The screening can be conducted as follows, for example, using themammalian One-Hybrid System (see Example 8).

First, a vector which expresses a protein of the present invention as afusion protein with a known peptide having DNA binding activity, such asthe DNA binding domain of GAL4 (designated as “primary vector”), and avector wherein a reporter gene is linked in an expressible mannerdownstream of the sequence to which the peptide with the DNA bindingactivity binds (designated as “secondary vector”) are constructed. Theprimary and secondary vectors are introduced into the cells and thereporter activities are detected. Examples of cells to be used in themethod include MG63, HeLa, and 293. In the secondary vector, anappropriate promoter (e.g., CMV promoter), whose transcriptionalactivity regulated by the protein of the present invention is to bedetected, can be used. The reporter activity detected by theintroduction of the primary and secondary vectors is compared with thatobtained without the primary vector. When the reporter activity changesupon the introduction of the vector as compared with that observed inthe absence of the primary vector, the change is evaluated as the“transcriptional regulation activity” of the protein of the presentinvention.

Next, the reporter activity is detected, in a similar manner, exceptcontacting the cells retaining the primary and secondary vectors with atest sample. The samples to be tested are not limited, but may be cellextracts, culture supernatants, fermented products of microorganisms,extracts of marine organisms, plant extracts, purified or partiallypurified proteins, peptides, non-peptide compounds, synthetic lowmolecular compounds, or natural compounds. Further, the test samples canbe compounds isolated by the screening for a compound that binds to aprotein of the present invention described above. Because of thedetection, a test sample can be determined as a compound that controlsthe transcriptional regulation activity of a protein of the presentinvention, when the “transcriptional regulation activity” of the proteinof the present invention increases or decreases in accordance with thecontact of the test sample.

Compounds that can be isolated by the screening method of this inventionmay be applied to treatment of diseases caused by expressional orfunctional abnormalities of a protein of this invention, or diseasesthat may be treated by regulating the activity of a protein of thisinvention. Compounds isolated by the screening method of this invention,wherein the structure of compounds is partially altered via addition,deletion, and/or replacement, are also included as compounds that bindto a protein of this invention.

When a protein or peptide of the present invention, or a compound thatcan be isolated by the screening method of the present invention is usedas a pharmaceutical for humans and other animals, such as, mice, rats,guinea pigs, rabbits, chicken, cats, dogs, sheep, pigs, bovines,monkeys, baboons, chimpanzees, the isolated compound can be administerednot only directly, but also as dosage forms using known pharmaceuticalpreparation methods. For example, according to the need, the drugs canbe taken orally as sugarcoated tablets, capsules, elixirs andmicrocapsules; or non-orally in the form of injections of sterilesolutions or suspensions with water or any other pharmaceuticallyacceptable liquid. For example, the compounds can be mixed withpharmacologically acceptable carriers or medium, specifically,sterilized water, physiological saline, plant-oil, emulsifiers,suspending agent, surface-active agent, stabilizers, flavoring agents,excipients, vehicles, preservatives and binders, into a unit dose formrequired for generally accepted drug implementation. The amount ofactive ingredient in these preparations makes a suitable dosage withinthe indicated range acquirable.

Examples of additives which can be incorporated into tablets andcapsules are: binders such as gelatin, corn starch, tragacanth gum andgum acacia; excipients such as crystalline cellulose; swelling agentssuch as corn starch, gelatin and alginic acid; lubricants such asmagnesium stearate; sweeteners such as sucrose, lactose or saccharin;flavoring agents such as peppermint, Gaultheria adenothrix oil andcherry. When the unit dosage form is a capsule, a liquid carrier such asoil can also be included in the above ingredients. Sterile compositesfor injection can be formulated following normal drug implementationsusing vehicles such as distilled water used for injections.

Physiological saline, glucose, and other isotonic liquids includingadjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodiumchloride, can be used as aqueous solutions for injections. These can beused in conjunction with suitable solubilizers, such as alcohol,specifically ethanol; polyalcohols such as propylene glycol andpolyethylene glycol; and non-ionic surfactants such as Polysorbate 80(TM) and HCO-50.

Sesame oil or Soy-bean oil can be used as a oleaginous liquid and may beused in conjunction with benzyl benzoate or benzyl alcohol assolubilizers. They further may be formulated with a buffer such asphosphate buffer and sodium acetate buffer, a pain-killer such asprocaine hydrochloride, a stabilizer such as benzyl alcohol and phenol,and an anti-oxidant. The prepared injection may be filled into asuitable ampule.

Methods well known to one skilled in the art may be used to administerthe pharmaceutical compounds of the present invention to patients, forexample as intra-arterial, intravenous, or percutaneous injections andas intranasal, transbronchial, intramuscular percutaneous, or oraladministrations. The dosage varies according to the body-weight and ageof a patient and the administration method, but one skilled in the artcan suitably select them. If the compound can be encoded by a DNA, theDNA can be inserted into a vector for gene therapy to perform thetherapy. The dosage and method of administration vary according to thebody-weight, age, and symptoms of a patient, but one skilled in the artcan select them suitably.

Although there are some differences according to the subject, subjectorgan, symptoms, and administration method, the dose of a protein of thepresent invention to be injected to a normal adult (weight 60 kg) is,for example, about 100 μg to about 20 mg per day.

Although there are some differences according to the symptoms, the doseof a compound that binds with a protein of the present invention andregulates the activity of the protein is considered to be about 0.1 mgto about 100 mg per day, preferably about 1.0 mg to about 50 mg per day,and more preferably about 1.0 mg to about 20 mg per day, whenadministered orally to a normal adult (weight 60 kg).

When administering parenterally in the form of an injection to a normaladult (weight 60 kg), although there are some differences according tothe patient, target organ, symptoms and method of administration, it maybe convenient to intravenously inject usually a dose of about 0.01 mg toabout 30 mg per day, preferably about 0.1 to about 20 mg per day, andmore preferably about 0.1 to about 10 mg per day. In addition, in thecase of other animals too, it is possible to administer an amountconverted to 60 kgs of body-weight or an amount converted to bodysurface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the eDNA sequence and amino acid sequence of murine 285(m285), SEQ ID NO: 1 and SEQ ID NO:2, respectively. Three zinc fingersare indicated in italic, and proline-rich region and alanine-rich regionare underlined with solid and dotted lines, respectively.

FIG. 2 depicts the comparison of the amino acid sequences of murine 285(SEQ ID NOS:27-44 and the three zinc finger domains of the Sp familytranscription factors and Sp family-like transcription factors. In thefigure, amino acid residues identical to those of Spi are indicated as“-”. The zinc finger domains are underlined and conserved cysteine andhistidine residues are shaded in gray.

FIG. 3 depicts the comparison of the N-terminal amino acid sequences ofmurine 285 (SEQ ID NO: 48), murine Sp1 (SEQ ID NO:45), human Sp2 (SEQ IDNO:46), and human Sp4 (SEQ ID NO:47). Amino acid residues conserved inthree of the four molecules are shown in gray, and those conserved inall molecules are indicated by reversed characters.

FIG. 4 depicts photographs demonstrating the expression of m285 in CL6by RT-PCR upon the induction of myocardial differentiation.DMSO-unstimulated cells are designated as 0 day.

FIG. 5 depicts photographs demonstrating the tissue distribution of m285expression by murine Northern blot analysis.

FIG. 6 depicts the cDNA sequence and amino acid sequence of human 285(h285), SEQ ID NO: 3 and SEQ ID NO: 4, respectively. The three zincfingers are indicated in italic, and the proline-rich region andalanine-rich region are underlined with solid and dotted lines,respectively.

FIG. 7 depicts the comparison of amino acid sequences of murine andhuman 285, SEQ ID NO: 2 and SEQ ID NO: 4, respectively. The three zincfingers and proline-rich regions are underlined with waved and solidlines, and the alanine-rich regions are boxed. They share a homology of97.5% at the amino acid sequence level.

FIG. 8 depicts photographs demonstrating intracellular localization ofm285 in CL6 cells.

FIG. 9 depicts an electrophoretogram of the binding assay between-GSTfusion 285F, 285N, and 285C to the 33-P labeled GC-box DNA probes.Unlabeled ds oligo was used as the competitor.

FIG. 10 depicts schematic illustrations of the activation assay andrepression assay.

FIG. 11 depicts a graph demonstrating the result of the activationassay. None of the tested plasmids activated the E1B minimal promoter.

FIG. 12 depicts a graph demonstrating the result of the repressionassay. 285F repressed CMV promoter activity in a dose dependent manner.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be explained in detail below with referenceto the following Examples, however, it is not construed as being limitedthereto.

EXAMPLE 1 Isolation of m285 Gene Fragment

Differentiation of undifferentiated murine embryonal carcinoma cellline, P19, to various neuronal cells, smooth muscle cells,cardiomyocytes, and such can be induced by stimulation with variousstimuli (DMSO, retinoic acid, etc.) (Bain G. et al. (1994) Bioessays 16,343-348; McBurney M W. (1993) Int. J. Dev. Biol. 37, 135-140). CL6 cellis an undifferentiated cell line established from P19 (Habara-Ohkubo A.(1996) Cell Struct. Funct. 21, 101-110). The CL6 cells can beefficiently differentiated to cardiomyocyte-like cells by adding 1% DMSOto the cell culture media under an adherent condition, while P19 must bemaintained in a floating condition to form an emryoid body for theinduction of differentiatiation. The CL6 cells express cardiac musclegene, such as cardiac α and β-HMC, approximately 8 days after thestimulation by 1% DMSO, and start a synchronized pulsation approximately10 days after the stimulation. The present inventors searched for genesinvolved in cardiac muscle differentiation as described below using thedifferentiation induction system of CL6 cells.

The culture of CL6 cells was carried out according to the method ofHabara-Ohkubo. Cells before and 4 days after adding 1% DMSO were treatedwith trypsin, were collected, and total RNA was obtained using RNeasytotal RNA isolation kit (QIAGEN). PolyA+ RNA was obtained from total RNAusing Mini-Oligo (dT) Spin Column Kit (5prime-3prime, Inc.) The obtainedpolyA+ RNA samples from CL6 cells before (A) and 4 days after (B) addingwith 1% DMSO were used as templates of the subtraction PCR method. Itwas conducted by subtracting (A) from (B), using CLONTECH PCR selectcDNA Subtraction Kit (CLONTECH) according to the manufacturer'sinstruction. The obtained subtracted PCR products were subcloned intopCR Blunt II vector using Zero blunt TOPO PCR Cloning Kit (invitrogen).Colonies retaining recombinants were amplified by colony-PCR describedbelow, and the nucleotide sequences were confirmed using nested primer 1(nP1) and nested primer 2R (nP2R) (CLONTECH). As a result, many genefragments whose expression increases in accordance with the induction ofdifferentiation to cardiomyocyte were obtained. Among these obtainedgene fragments, fragment PS40-285 was further analyzed (see, Example2-).

Colony PCR of the present Examples was carried out as follows. Coloniesretaining the recombinants were directly suspended in 20 μl PCR reactionmixture comprising SPORT FW (5′-TGT AAA ACG ACG GCC AGT-3′/SEQ ID NO:5), SPORT RV (5′-CAG GAA ACA GCT ATG ACC-3′/SEQ ID NO: 6), and KOD dashpolymerase; and PCR was carried out at a condition of 94° C. for 1minute, and 32 cycles of 96° C. for 15 seconds, 55° C. for 5 seconds,and 72° C. for 25.seconds. The amplified PCR products were confirmed byagarose gel electrophoreses and purified as needed using MicrospinS-300, S-400 gel filtration (Pharmacia) or using Multiscreen HV plate(Millipore)+BioGel-P60 (BioRad) to prepare templates for the sequencingreaction.

Determination of nucleotide sequences of the present Examples wascarried out as follows. PCR products of colony PCR, RT-PCR, and suchwere used as the templates of a sequencing reaction. After the PCR,products were confirmed by agarose gel electrophoreses, and theobjective PCR products were either cut out from the gel when impuritieswere present, or those without contaminants were purified by gelfiltration. Cycle sequencing using BigDye Terminator Cycle Sequencing FSready Reaction Kit (Perkin-Elmer) was carried out as the sequencingreaction. Unreacted primers, nucleotides, and such were removed by 96well precipitation HL kit (AGTC), and the nucleotide sequences weredetermined using ABT 377 or ABI 377XL DNA Sequencer (Perkin-Elmer).

EXAMPLE 2 cDNA Cloning of m285 Gene

Using mouse 10.5 dpc embryo cDNA plasmid library as a template, cDNAcontaining total ORF of m285 was cloned based on the nucleotide sequenceof a gene fragment PS40-285 (FIG. 1). The nucleotide sequence of thecloned m285 cDNA containing total ORF was determined. Determination ofthe nucleotide sequence was carried out as follows.

According to the nucleotide sequence of the subtraction clone PS40-285,primers, 285-A (5′-CAG CCC TGG GTA AAA TGT AAG TC-3′/SEQ ID NO: 7),285-B (5′-TCG AGG TAG CTG ACA AAG AGT AAC-3′/SEQ ID NO: 8), 285-C(5′-TCA CCA GTG CAG GGA TCT ACA AA-3′/SEQ ID NO: 9), and 285-D (5′-GCAGTC AGG TGT CTT GGT CTG ATT-3′/SEQ ID NO: 10), were synthesized onBeckman automatic DNA synthesizer. SuperScript Mouse 10.5 day embryocDNA library (GIBCO) was added to 5 ml LB-Amp medium at a concentrationof 2.1×10³ clones, cultured at 30° C., and then plasmids were obtainedusing QIAspin mini prep kit (QIAGEN). Using the plasmids as a template,reaction by Advantage cDNA polymerase (CLONTECH) was carried out withfollowing primers and cycles.

Specifically, following primers were used for the reaction: 285-A+SPORTFW (1st. 3′ RACE) and 285-C+SPORT T7 (5′-TAA TAC GAC TCA CTA TAGGG-3′/SEQ ID NO: 11) (nested 3′ RACE) as 3′ RACE primers, and285-B+SPORT RV (1st. 5′ RACE) and 285-D+SP6 (5′-ATT TAG GTG ACA CTA TAGA-3′/SEQ ID NO: 12) (nested 5′ RACE) as 5′ RACE primers. The first RACEwas carried out at a condition of 95° C. for 1 minute, and 30 cycles of96° C. for 15 seconds, 63° C. for 15 seconds, and 68° C. for 3 minutes;and the nested RACE was carried out under the same conditions exceptthat the cycle number was 15. The nucleotide sequences of nested RACEproducts were determined using primers that were used for the RACE. Asfor the 5′ RACE products, the nucleotide sequences were determined using285-E (5′-TTC TCG CCC GTG TGA GTC CGC A-3′/SEQ ID NO: 13), and 285-F(5′-TCC AGA CTT TTC CAC CCT TGG ACT-3′/SEQ ID NO: 14).

As a result, m285 was revealed to comprise 1830 bases that encode 398amino acids. The amino acid sequence contained three zinc finger domainsin its C-terminus, and a proline-rich rich and an alanine-rich region inthe middle of the sequence. (FIG. 1).

Next, BLAST database search of the m285 cDNA was conducted, whichrevealed that it has a high homology with a human UniGene clone (NCBIhuman UniGene clone No.: Hs.145921) and a human HTG clone (GenBankaccession No.: AC007405), and thus it was discovered that m285 is themouse counterpart of these clones. However, no known gene with highhomology covering the whole gene could be found in the database; andthus, it was determined to be a novel gene. Because of database searchwith the amino acid sequence of m285, three zinc finger domains thatwere located in the C-terminus showed very high homology with the Spfamily transcription factors (FIG. 2). Among these factors, Sp4 showedan especially high homology, which reached 88.6% identity in the threezinc finger domains. In addition, in the N-terminus, it had a completelyidentical region (though only 11 amino acid residues) to Sp1, Sp2, andSp4 (FIG. 3). However, other regions besides those described above didnot show homology to the Sp family genes. Further, no known gene withhigh homology covering the whole m285 sequence could be found; thus m285was revealed to be a novel gene with three zinc finger domains.

EXAMPLE 3 m285 Gene Expression in CL6 Differentiation Induction System

CL6 is a cell line isolated from undifferentiated embryonal carcinomacell line, P19, and upon differentiation induction by DMSO, itdifferentiates from the undifferentiated character to acardiomyocyte-like cell in approximately 10 days. m285 was obtained as agene whose expression increases in accordance with the induction ofcardiomyocyte differentiation of CL6 (4 days after DMSO stimulation)Therefore, to analyze the m285 gene expression in the CL6differentiation induction system by RT-PCR, RNAs were isolated fromundifferentiated CL6 (without the differentiation induction with DMSO),and CL6 cells 4 days, 8 days, and 12 days after the differentiationinduction with DMSO. At the same time, RNA isolated from adult mouseheart was also analyzed. The RT-PCR was carried out as follows.

Using the total RNA prepared from CL6 cells before addition of DMSO, andcells 4 days, 8 days, and 12 days after the addition of 1% DMSO, andmouse heart total RNA of C3H/He (Nippon Gene); SuperScript II (GIBCO) asRTase; and (dT)₃₀VN primers, cDNAs, which are used as templates ofRT-PCR, were synthesized. The RT-PCR with Advantage cDNA polymeraseusing 285-A and 285-B primer sets and, as a control, G3PDH 5′ (5′-GAGATT GTT GCC ATC AAC GAC C-3′/SEQ ID NO: 15) and G3PDH 3′ (5′-GTT GAA GTCGCA GGA GAC AAC C-3′/SEQ ID NO: 16) primer set was carried out at acondition of 95° C. for 1 minute, and 28 cycles (20 cycles for G3PDH) of96° C. for 15 seconds, 60° C. for 15 seconds, and 68° C. for 30 seconds.

Because of the RT-PCR, m285 was revealed to be transiently expressed inonly CL6 cells 4 days after the differentiation induction with DMSO(FIG. 4). No expression was observed in CL6 cells before differentiationinduction, those eight or more days after the differentiation induction,and in adult mouse heart.

EXAMPLE 4 Tissue Distribution of m285 Gene Expression

Next, m285-expressing tissues were searched by Northern blot analyses.PCR with 285-Eand285-Fusing the 5′ RACE products of Example 3 as atemplate was carried out to obtain a product of approximately 0.9 kb.The PCR product was labeled with [α-³²P]dcTp using Megaprime DNAlabelling system (Amersherm), and then unreacted [α-³²p]dCTP was removedto prepare m285 probe. Using Mouse Multiple Tissue Northern (MTN) blotand Mouse Embryo MTN blot (CLONTECH), hybridization was carried out at68° C. in ExpressHyb Hybridization Solution (CLONTECH) according to themanufacturer's instruction. After exposing to imaging plates, analyseswere accomplished using BAS2000 imaging analyzer.

As a result, in the prenatal period, a very high expression of m285 wasobserved in 17 dpc murine embryo; and little expression is observed in 7dpc, 11 dpc, and 15 dpc embryos. In the adult mice, expression intestis, and weak expression in brain, heart, liver, and kidney tissueswere detected (FIG. 5). Considering the result with the above-describedexpression patterns in CL6 differentiation induction system, m285 isexpected to be somehow involved in the late stage of development,because of its very high expression in 17 dpc embryo.

EXAMPLE 5 cDNA Cloning of h285 Gene

Based on the nucleotide sequence of m285, and the above-mentioned humanUniGene clone (Hs.145921; NCBI human UniGene clone number) and human HTGclone (AC007405; GenBank accession number), cloning of h285 gene wascarried out as follows.

Using human fetal brain Marathon ready cDNA (CLONTECH) as a template,RACE was carried with either 285-J (5′-CTT TGC (A/G)CA GTA CCA GAG CCAGAT-3′/SEQ ID NO: 17) and AP1 (1st. 3′ RACE), or 285-hC (5′-AAG AAG AAGCAG CAC GTG TGC CAC-3′/SEQ ID NO: 18) and AP2 (nested 3′ RACE) usingAdvantage cDNA polymerase following procedures described below.Specifically, the first RACE was carried out under a condition of 95° C.for 1 minute, and 35 cycles of 96° C. for 15 seconds, 63° C. for 15seconds, and 68° C. for 2 minutes; and nested RACE was carried out underthe same condition except for the cycle number being 20.

Using rTaq (TaKaRa), dA was added to the nested RACE products tosubclone the products into pCR 2.1 TOPO (invitrogen), and determinetheir nucleotide sequences.

RT-PCR with PCRx system-Platinum Taq (GIBCO) using human fetal braincDNA (CLONTECH) as a template, and 28.5-hG (5′-GGC GTC CCG CTC CGC AGCCA-3′/SEQ ID NO: 19) and 285-hB (5′-CCG GCC TCA GCG ACT TTG AGCTT-3′/SEQ ID NO: 20) as primers was carried out under a condition of 95°C. for 1 minute, and 40 cycles of 96° C. for 15 seconds, 58° C. for 15seconds, and 72° C. for 1.5 minutes.

Following the addition of dA to the RT-PCR products using rTaq (TaKaRa),the products were subcloned into pCR 2.1 TOPO (invitrogen) to determinetheir nucleotide sequences (FIG. 6).

As a result, the obtained h285cDNA, as same as m285, was considered toencode 398 amino acids. According to a comparison with m285, h285 showed91.1% and 97.5% homology at the nucleotide sequence level within thecoding region and the amino acid sequence level, respectively (FIG. 7).The cDNA sequence was almost identical to human UniGene clone(Hs.145921) and human HTG clone (AC007405) (these sequences containseveral bases of addition/deletion, which seem to be the result ofsequencing errors). The amino acid sequence of h285 comprised, similarlyto m285, three zinc finger domains in its C-terminus that share a veryhigh homology to those of the Sp family, N-terminal 11 amino acidresidues completely identical to those in Sp1, Sp2, and Sp4, andproline-rich and alanine-rich regions in the middle (FIG. 7).

No known gene with high homology to h285 covering the whole region couldbe found as in the case of m285, and h285 was revealed to be a novelgene.

EXAMPLE 6 Intracellular Localization of m285

Generally, transcription factors diversely regulate gene expressions bybinding to DNA. Although some transcription factors exist that usuallyexist in the cytosol, translocate to the nucleus by certainsignal/stimuli, and regulate transcription, many of the transcriptionfactors including the Sp family transcription factors localize in thenucleus of the cell. Therefore, intracellular localization of the clonedm285 was examined.

Plasmids that express full-length m285 (285F; amino acid residues 1 to398), N-terminal region of m285 (285N; amino acid residues 1 to 278),and C-terminal region of m285 (285C; amino acid residues 271 to 398) asEGFP fusion proteins were constructed. 285N comprises the N-terminal 11amino acid residues that are identical to those of Sp1, Sp2, and Sp4, aproline-rich region and an alanine-rich region; whereas 285C comprisesthe C-terminal three zinc finger domains that share a very high homologyto those of the Sp family. Plasmid constructions were carried out asfollows. First, 285EcoRI-N (5′-GAA TTC CCT TCA AGC AGT AGC CAT GGCCG-3′/SEQ ID NO: 21), 285EcoRI-P (5′-GAA TTC CTT TGC ATA CCA GAG CGAGAT-3′/SEQ ID NO: 22), 285SalI-K (5′-GTC GAC ATC TGG CTC TGG TAC TGT GCAAAG-3′/SEQ ID NO: 23), and 285SalI-M (5′-GTC GAC AGT GTC CCG GTG CGC TCATAG GTC-3′/SEQ ID NO: 24), derived from m285 were synthesized. Then,cDNA was synthesized from total RNA that was isolated from CL6 cells 4days after DMSO stimulation, and RT-PCR was carried out using285EcoRI-N+285SalI-M (285F), 285EcoRI-N+285SalI-K (285N), and285EcoRI-P+285SalI-K (285C). Following the addition of dA-to the RT-PCRproducts with rTaq (TaKaRa), the products were subcloned into pCR 2.1TOPO, respectively.

pCR2.1-285F, 285N, and 285C were digested with EcoRI and SalI, andobtained fragments were subcloned into pGEX 4T-3 (Pharmacia) in frame.JM-109was transformed with the plasmids and GST fusion proteins(GST-285F, GST-285N, and GST-285C) were obtained following themanufacturer's protocol. The fusion proteins were subjected to DNAbinding assay described below.

Similarly, EcoRI-SalI fragments of pCR2.1-285F, 285N, and 285C weresubcloned into pEGFP-C1 (CLONTECH) in frame (pEGFP-285F, pEGFP-285N, andpEGFP-285C).

Next, thus constructed EGFP fusion protein expression plasmids weretransfected into CL6 cells using FuGENE6 (Roche Diagnostics) accordingto the manufacturer's protocol for transient expression of the fusionproteins; and after 48 hours, cells were observed under fluorescencemicroscopy. At the same time, a plasmid that expresses EGFP alone wasalso transfected in the same way.

As a result of the observation using fluorescence microscopy,fluorescence was observed from the whole cells that were transfectedwith EGFP alone or EGFP-285N, while the fluorescence could be onlydetected in the nucleus for those transfected with EGFP-285F andEGFP-285C (FIG. 8). Thus, m285 was revealed to be a protein localized inthe nucleus, which nuclear translocation of the protein requires itsC-terminal region including three zinc finger domains.

EXAMPLE 7 DNA Binding Activity

Sp1 is known to bind to a GC-box through its three zinc finger domains.The m285 cloned in the present invention has three zinc finger domainsthat share a very high homology with the Sp family, and therefore theprotein was expected to bind to a GC-box. Thus, 285F, 285N, and 285Cwere expressed as GST fusion proteins in E. coli to examine theirbinding to a GC-box. GST fusion proteins purified with glutathioneSepharose, and as the GC box a DNA probe having the nucleotide sequencein the Waf-1 promoter region which sequence binds to Sp1 were used. DNAprobe was prepared by labeling ds-(5′-TCG AAA GGA GGC GGG ACC CGAGCT-3′/SEQ ID NO: 25) containing a GC-box sequence with 33-P. The DNAprobe was reacted with GST-285F, GST-285N, and GST-285C described abovein EMSA buffer (20 mM HEPES, 40 mM KCl, 6 MM MgCl₂, 1 mM EGTA, 1 mM DTT,0.1% NP-40, 10% Glycerol, 0.15% BSA, 25 ng/μl sonicated salmon spermDNA) for 20 minutes. To examine the presence of competitive inhibition,either an unlabeled GC-box DNA probe or unlabeled mutant GC-box DNAprobe (ds-(5′-TCG AAA GGA GTT TTG ACC CGG AGC T-3′/SEQ ID NO: 26)) wasfurther added to a similar reaction system. Recombinant Sp1 was used asa positive control. The reactants were electrophoresed on 5% acrylamidegel and were exposed to imaging plates. Analyses were performed usingBAS2000 imaging analyzer.

As a result of gel shift assay, bands that were considered to correspondto DNA-protein complex were observed when GST-285F, GST-285C, orrecombinant Sp1 were reacted with the 33-P labeled GC-box DNA probe(FIG. 9; lanes 1, 4, and 9). No complex b and could be observed forGST-285N reacted with the GC-box DNA probe (FIG. 9; lane 7). Theaddition of unlabeled GC-box DNA probe vanished or decreased the complexbands of GST-285F, GST-285C, or recombinant Sp1, and the GC-box DNAprobe (FIG. 9; lanes 2, 5, and 10). Further, the addition of unlabeledmutant GC-box DNA probe to a similar reaction system had no effect onthe complex bands of GST-285F, GST-285C, or recombinant Sp1, and theGC-box DNA probe (FIG. 9; lanes 3, 6, and 11). These results indicatethat m285binds specifically to a GC-box sequence through its C-terminalregion comprising three zinc finger domains.

EXAMPLE 8 Transcriptional Regulation by Mammalian One-Hybrid System

The Sp family transcription factors are known to enhance transcriptionthrough their glutamine-rich regions; however, transcriptionalrepression through regions other than glutamine-rich region has alsobeen reported for Sp1 and Sp3. Thus, the mechanism of thetranscriptional regulation (activation/repression) by the newly cloned285 was examined by the mammalian One-Hybrid System (FIG. 10).

Vectors expressing the Gal4 DNA binding domain and 285F, 285N, or 285Cas fusion proteins were constructed by subcloning EcoRI-SalI fragmentsof pCR2.1-285F, 285N, and 285C into pM (CLONTECH) in frame (pM-285F,pM-285N, and pM-285C). They were used as assay plasmids.

Activation assay was performed using pG5-Luc (5× Gal4 binding site—E1Bminimal promoter—Luciferase; Sowa Y. et al. (1999) Cancer Res. 59:4266-4270) and pM-Sp1 (Gal4 DNA binding domain —Sp1; Sowa Y. et al.(1999) Cancer Res. 59: 4266-4270) as a reporter plasmid and positivecontrol plasmid, respectively. Repression assay was performed usingpKO-114 (5×Gal4 binding site—CMV promoter—Luciferase) as a reporterplasmid. pKO-114 was constructed by digesting pGL3-basic vector(Promega) with SmaI and HindIII, and then inserting into the site 5×Gal4binding site (SmaI-XbaI fragment) derived from pG5-Luc and CMV promoterderived from pcDNA3.1-HisA (Invitrogen) (SpeI-HindIII fragment).pRL/SV40 (Promega) was used as an internal control plasmid in bothassays.

MG63 cells (obtained from Kyoto Prefectural University) were seeded onto12 well plates at 8×10⁴ cells/well and cultured. On the next day, MG63cells were transfected with the assay plasmid (or positive controlplasmid), reporter plasmid, and internal control plasmid, usingSuperfect (QIAGEN). After 2 days, the luciferase activities weremeasured using Dual-Luciferase Reporter Assay System (Promega) Accordingto the result of the activation assay (FIG. 10),it was revealed that285does not activate the transcription of CMV minimum promoter in MG63cells (FIG. 11). To the contrary, the repression assay (FIG. 10)revealed that 285 represses the transcriptional activity of CMV promoterin MG63 cells (FIG. 12). The level of the repression was almost equal tothat of KRAB (derived from KOX) used as a control.

Thus, it was revealed that 285 functions as a transcription factor.

INDUSTRIAL APPLICABILITY

The present invention provides genes encoding a novel transcriptionfactor that is considered to belong to the Sp1 transcription factorfamily. Proteins encoded by such genes are expected to regulate thetranscription of genes by binding to a GC-box through their zinc fingerdomains. The genes and proteins of the present invention are useful astargets for developing therapeutic agents.

1. An isolated mammalian DNA selected from the group consisting of: (a)a DNA encoding a protein comprising the amino acid sequence of SEQ IDNO: 2or 4; (b) a DNA containing the full length coding region of thenucleotide sequence of SEQ ID NO: 1 or 3; (c) a DNA encoding a proteincomprising the amino acid sequence that has a sequence homology of 95%or more to the entirety of the amino acid sequence of SEQ ID NO: 2 or 4;and wherein the protein represses the transcriptional activity of a CMVpromoter and has binding activity to a GC-box of the Waf-1 promoter; (d)a DNA encoding a protein fragment comprising the amino acid sequence ofresidues 271 to 398 of SEQ ID NO: 2; and (e) a DNA consisting ofnucleotides 811-1194 of SEQ ID NO:
 3. 2. A recombinant vector into whichthe DNA of claim 1 is inserted.
 3. An isolated transformed cell modifiedto contain the DNA of claim
 1. 4. A method for producing a protein orprotein fragment thereof which is a transcription factor that repressesa CMV promoter wherein the method comprises the steps of culturing atransformed cell modified to contain an isolated mammalian DNA selectedfrom the group consisting of: (a) a DNA encoding a protein comprisingthe amino acid sequence of SEQ ID NO: 2 or 4; (b) a DNA containing thefull length coding region of the nucleotide sequence of SEQ ID NO: 1 or3; (c) a DNA encoding a protein comprising the amino acid sequence thathas a sequence homology of 95% or more to the entirety of the amino acidsequence of SEQ ID NO: 2 or 4; and wherein the protein represses thetranscriptional activity of a CMV promoter and has binding activity to aGC-box of the Waf-1 promoter; (d) a DNA encoding a protein fragmentcomprising the amino acid sequence of residues 271 to 398 of SEQ ID NO:2; and (e) a DNA consisting of nucleotides 811-1194 of SEQ ID NO: 3,expressing a protein or protein fragment encoded by the DNA of any of(a)-(e) contained within said transformed cell, and recovering theexpressed protein or protein fragment from said transformed cell or aculture supernatant thereof.
 5. An isolated DNA encoding a proteincomprising the amino acid sequence of SEQ ID NO: 2 or
 4. 6. An isolatedDNA containing the full length coding region of the nucleotide sequenceof SEQ ID NO: 1 or 3.